Abstract
Genetically modified swine disease models are becoming increasingly important for studying molecular, physiological and pathological characteristics of human disorders. Given the limited history of these model systems, there remains a great need for proven molecular reagents in swine tissue. Here, to provide a resource for neurological models of disease, we validated antibodies by immunohistochemistry for use in examining central nervous system (CNS) markers in a recently developed miniswine model of neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant tumor predisposition disorder stemming from mutations in NF1, a gene that encodes the Ras-GTPase activating protein neurofibromin. Patients classically present with benign neurofibromas throughout their bodies and can also present with neurological associated symptoms such as chronic pain, cognitive impairment, and behavioral abnormalities. As validated antibodies for immunohistochemistry applications are particularly difficult to find for swine models of neurological disease, we present immunostaining validation of antibodies implicated in glial inflammation (CD68), oligodendrocyte development (NG2, O4 and Olig2), and neuron differentiation and neurotransmission (doublecortin, GAD67, and tyrosine hydroxylase) by examining cellular localization and brain region specificity. Additionally, we confirm the utility of anti-GFAP, anti-Iba1, and anti-MBP antibodies, previously validated in swine, by testing their immunoreactivity across multiple brain regions in mutant NF1 samples. These immunostaining protocols for CNS markers provide a useful resource to the scientific community, furthering the utility of genetically modified miniswine for translational and clinical applications.
Highlights
Animal models are essential tools for studying the underlying mechanisms of disease as well as providing a platform for preclinical research and drug discovery
The following markers (GFAP, IBA1, and myelin basic protein (MBP)) have been validated in the cerebrum of wildtype swine previously [23]. These antibodies have not been validated in other areas of the brain that are commonly affected in neurological diseases
GFAP, a marker of intermediate filaments in astrocytes that become hypertrophic in response to insult, has been shown to be increased in a number of neurological diseases, including neurofibromatosis type 1 (NF1) [24]
Summary
Animal models are essential tools for studying the underlying mechanisms of disease as well as providing a platform for preclinical research and drug discovery. The mouse brain lacks gyri and sulci in the cerebrum and has much less white matter [7]; physiologically, mice differ in immune receptors, cell types, and signaling pathways [8] These anatomical and physiological differences found in the rodent systems cannot recapitulate human disease. Successful genetically modified miniswine models have been established to study a number of human diseases including atherosclerosis, cancer, ataxia telangiectasia, cystic fibrosis, and neurofibromatosis type 1 [3, 6, 9, 10] While these large animal systems can better recapitulate many of the hallmarks of human disease, there are limited tools and reagents that have been well described and validated in these models, limiting the translatable application of these models. To provide this resource to the scientific community, we validate a number of antibodies relevant to the study of the brain and neurological disorders using immunohistochemistry in porcine brain tissue
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